Project Summary/Abstract Dysfunctions in one carbon (1C), or folate, metabolism are well-known for their deleterious effects on human development, causing neural tube defects. However, the pathway is also implicated in both mitochondrial dysfunction (found in aging as well as rare, genetic disorders), and many cancers. Because of the centrality of 1C metabolism across these diverse human diseases, it is already the target of drugs such as methotrexate. Nevertheless, the underlying biochemical logic of the pathway remains incredibly complex, rendering its fundamental functions difficult to understand, and therefore limiting its ability to be targeted by further drugs. Contributing to the complexity of 1C metabolism are its subcellular compartmentalization and redox dependency. It is not well appreciated that there are two parallel branches of 1C metabolism, one in the cytosol and one in the mitochondria, which are controlled by the subcellular redox state of NADH and NADPH cofactors. Therefore, to fully characterize the biochemical logic driving 1C metabolism, it is necessary to have tools to precisely perturb the subcellular redox state of these cofactors. Recent work by the Mootha laboratory has provided tools to do exactly that: a collection of four water-forming oxidases (NOXes) that can selectively oxidize the NADH or NADPH pool in the cytoplasm or the mitochondria. This proposal aims to use these powerful genetic tools to decipher, for the first time, the biochemical logic underlying 1C metabolism in two states of cellular stress: mitochondrial dysfunction and hypoxia. These perturbations are good models to probe the activity of 1C metabolism. Mitochondrial dysfunction upregulates the pathway, and simultaneously reduces both the mitochondrial and cytosolic NADH pools. Hypoxia has also been shown to significantly remodel the mitochondrial 1C branch and additionally produces cytotoxic reactive oxygen species (ROS). To better characterize the complex interactions between 1C metabolism, subcellular redox state, and ROS, this proposal will leverage high-resolution mass spectrometry to measure whole-metabolome perturbations. Finally, this proposal will couple the metabolomics dataset with measurements of cytotoxic reactive oxygen species and use an already-established cell line lacking a critical mitochondrial 1C enzyme to isolate the contributions of 1C on ROS. 1C metabolism plays a critical role in human development, cancer, and mitochondrial dysfunction. However, its underlying biochemical regulation remains poorly understood. Leveraging recently developed genetic tools to modulate subcellular redox homeostasis with high-resolution metabolomics, this proposal aims to decipher the biochemical logic of the 1C metabolic pathway with implications for current and pressing problems in human health and disease.